Interleukin-6 (IL-6) is a multi-functional cytokine playing a central role in host defense mechanisms. Heinrich et al., Biochem. J. (1990) 26.5:621; Van Snick, J. Annu. Rev. Immunol. (1990) 8:253; and Himno et al., Immunol. Today (1990) 11:443. However, in a variety of human inflammatory, autoimmune, and neoplastic diseases, abnormal IL-6 production is observed and has been suggested to play a role in the pathogenesis of those diseases. Hirano et al., supra; Sehgal, P. B., Proc. Soc. Exp. Biol. Med. (1990) 195:183; Grau, G. E., Eur. Cytokine Net (1990) 1:203; Bauer et al., Ann. Hematol. (1991) 62:203; Campbell et al., J, Clin, Invest. (1991) 7:739; and Roodman et al., J. Clin. Invest. (1992) 89:46. Inhibitors of IL-6 bioactivity might thus be useful to study its role in disease and could have broad therapeutic applications.
IL-6 overproduction is involved in sepsis (Starnes, Jr., H. F. et al., J. Immunol. (1990) 145:4185), and is also implicated in multiple myeloma disease, or plasma cell leukemia (Klein, B. et al., Blood (191) 78:1198). Other diseases include bone resorption (osteoporosis) (Roodman, G. D. et al., J., Clin, Invest. (1992) 89:46; Jilka, R. L. et al., Science (1992) 257:88-91), cachexia (Strassman, G. et al., J. Clin. Invest. (1992) 89:1681), psoriasis, mesangial proliferative glomerulonephritis, renal cell carcinoma, Kaposi's sarcoma, rheumatoid arthritis, hyper gammaglobulinemia (Grau, G. E. et al., J. Exp. Meal. (1990) 172:1505), Castleman's disease, IgM gammapathy, cardiac myxoma and autoimmune insulin-dependent diabetes (Campbell, I. L. et al., J, Clin, Invest., (1991) 87:739).
IL-6 functions through interaction with at least two specific receptors on the surface of target cells. Taga et al., J. Exp. Med. (1987) 166:967; and Coulie et al., Eur. J. Immunol, (1987) 17:1435. The cDNAs for these two receptor chains have been cloned, and they code for two transmembrane glycoproteins: the 80 kDa IL-6 receptor "IL-6R") and a 130 kDa glycoprotein called "gp130". Yamasaki et al., Science (1988) 241:825; and Hibi et al., Cell (1990) 63:1149. IL-6 interacts with these glycoproteins following a unique mechanism. First, IL-6R binds to IL-6 with low affinity (Kd=about 1 nM) without triggering a signal. Taga et al., Cell (1989) 58:573. The IL-6/IL-6R complex subsequently associates with gp130, which transduces the signal. Hibi et al., supra; and Taga et al., supra. Gp130 itself has no affinity for IL-6 in solution, but stabilizes the IL-6/IL-6R complex on the membrane, resulting in high affinity binding of IL-6 (Kd=about 10 pM). Hibi et al., supra. It was recently found that gp130 is also a low affinity receptor for oncostatin M and an affinity converter for the LIF receptor (Gearing, D. P. et al., Science (1992) 255:1434).
Mature human (h) IL-6 is a 185 amino acid polypeptide containing two disulfide bonds (Cys.sub.45 to Cys.sub.51 and Cys.sub.74 to Cys.sub.84). Clogston et al., Arch. BioChem. Biohphys. (1989) 272:144. The first 28 residues can be deleted without affecting bioactivity. Brakenhoff et al., J. Immunol, (1989) 143:1175. Bioactivity of hIL-6 appears to be conformation dependent. Large internal deletions disrupt the overall structure of the molecule and completely abolish activity. Snouwaert et al., J. Immunol. (1991) 146:585; and Fontaine et al., Gene (1991) 104:227. Maintenance of the second (but not the first) disulfide bond is critical, especially in bioassays involving human cell lines. Snouwaert et al., J, Biol. Chem. (1991) 266:23097. Regions critical to activity comprise residues Ile.sub.30 to Asp.sub.35 (see Brakenhoff et al., supra; Fontaine et al., supra; and Arcone et al., FEBS Letters (1991) 288:197), Ala.sub.154 to Thr.sub.164 (see Ida et al., Biochem. Biophys. Res. Commun. (1991) 165:728; and Nishimura et al., FEBS Letters (1991) 281:167) and Arg.sub.183 to Met.sub.185 (see Kr uttgen et al., FEBS Letters (1990) 26.2:323; Brakenhoff et al., J. Immunol. (1990) 145:561; and Kr uttgen et al., FEBS Letters (1990) 273:95). Substitution analysis of individual residues have implicated Leu.sub.159, Met.sub.162 and Leu.sub.166 to be important both for activity and binding to IL-6R (see Nishimura et al., supra). A positive charge and .alpha.-helical C-terminal structure were found to be essential for activity. L utticken et al., FEBS Letters (1991) 282:265.
One method for neutralization of IL-6 activity is the use of antibodies to IL-6. Neutralizing monoclonal antibodies (MAbs) to IL-6 can be divided in two groups, based on the recognition of two distinct epitopes on the IL-6 molecule, designated Site I and Site II. Site I is a conformational epitope composed of both amino terminal and carboxy terminal portions of the IL-6 molecule: the amino terminal portion includes amino acids Ile.sub.30 -Asp.sub.35 ; while the carboxy terminal portion includes critical amino acids Arg.sub.183 -Met.sub.185. Site II includes critical amino acids Ala.sub.154 -Thr.sub.163. Brakenhoff et al. (1990), supra.
Another way to neutralize IL-6 activity is to inhibit the ligand-receptor interactions with specific receptor-antagonists. The feasibility of this general type of approach was recently demonstrated with a natural occurring receptor antagonist for interleukin-1. Harmurn, C. H. et al., Nature (1990) 343:336-340. However, no natural receptor-antagonist has been identified for IL-6 so far. Nor has any hIL-6 variant with antagonistic properties been discovered. This invention uses the information gleaned from the Site I and Site II work with MAbs to construct hIL-6 variants that act as IL-6 receptor antagonists.